We will characterize the molecular mechanisms through which estrogen and progesterone regulate the expression of the prolactin gene. These studies will further our understanding of the interrelationships between these hormones in the control of normal growth, development, and reproductive function, and may provide insights into how estrogen and progesterone, through their effects on prolactin gene expression, contribute to the development of mammary cancer. We have shown that estrogen stimulates prolactin gene transcription through both direct and indirect pathways. In Specific Aim I, we will test the hypothesis that estrogen indirectly stimulates prolactin gene transcription by inhibiting the release of dopamine from the hypothalamus. This will be done directly by pituitary and the turnover of within the tuberoinfundibular neurons of the hypothalamus, and indirectly by examining the effects of estrogen on prolactin gene transcription in pituitaries that have been transplanted under the Kidney capsule. Our preliminary data support a physiological role of progesterone in the regulation of prolactin gene expression. In Specific Aim 2, we will examine in Vivo and in Vitro the regulation of prolactin gene expression by progesterone, and the interactions between progesterone and estrogen in this regulation. In these experiments the rate of the gene transcription will be quantified by a nuclear transcription assay, mRNA level by slot or northern blot procedures, and rate of prolactin synthesis by immunoprecipitation of pulse-labeled prolactin. In all experiments, the expression of the closely related growth hormone gene will also be examined as a control. If regulation by these steroid hormones is observed at the transcriptional level, the control elements which confer this responsiveness will be identified in experiments where various regions of the prolactin gene domain are linked to the bacterial CAT gene and the fusion genes are transfected into hormone-responsive cells. In Specific Aim 3, we will define the physiological roles of estrogen and progesterone in regulating the expression of the prolactin gene during the estrous cycle of the rat, and identify the levels at which these hormones function in this regulation. In these experiments, the rate of transcription of the prolactin gene, the level of prolactin mRNA and the rate of prolactin synthesis will be examined throughout the course of the estrous cycle and the effects of antiestrogens and antiprogestins on these parameters.